Identification of high affinity aptamers using massively parallel DNA sequencing

The vast majority of molecules are far too small to be seen with the unaided eye. In most cases the only way to detect them is with other molecules that fit round them like a key fits round a lock. The human body produces molecules like this called antibodies. When we are invaded by harmful bacteria antibodies stick to them and mark them out for destruction. Many years ago scientists realized that they could use antibodies to detect almost any kind of molecule. This opened the door to a whole range of new technologies in medicine, healthcare and research. DNA is familiar to most people as the molecule that contains the information for living things, but it can also fold into three-dimensional shapes that resemble antibodies. The shape of these structures depends on the sequence of information encoded in the DNA. Twenty years ago scientists speculated that it might be possible to combine the information properties of DNA with its ability to recognize other molecules. In a process that resembles natural selection they mixed many different DNA sequences with a single type of protein molecule. Most of them did not stick, but quite a few did, some of them more tightly than others. The scientists then used the information in DNA to make many copies of the sequences that stuck and mixed this amplified population with the protein again. This time some of the DNA that survived the first round of selection was excluded by sequences that stuck to the protein more tightly. These sequences were discarded while those that stuck were amplified to produce an even more enriched population. After many rounds of selection and amplification only a few sequences remained. The scientists called these surviving sequences aptamers after a Latin word that describes the way that other molecules fit into them like a key fits into a lock. Aptamers have many advantages over antibodies. They are smaller and more robust, and once the information encoded in an aptamer is known large amounts of it can be made inexpensively. With advantages like these it might be thought that aptamers would have supplanted antibodies long ago, but twenty years after their discovery they are still the poor-relation. The problem is that aptamers do not stick to other molecules as tightly as antibodies and recently scientists have found out why. The natural selection process used to identify them not only eliminates sequences that do not stick to the protein at all but also sequences that stick to it less strongly than the strongest. If these weaker sequences are joined to the strongest sequence a new aptamer is produced that sticks to the protein hundreds of times more tightly than the original. Technologies that read the information encoded in DNA are known as sequencing technologies. When aptamers were first discovered twenty years ago it required a great deal of effort to read the sequence of a single aptamer even though it contained less than a hundred bits of information. Now by contrast the entire 3 billion bits of information in the human genome can be read in only a few days. These advances have made it feasible to read the information encoded in all the DNA sequences that bind to a protein and not just the few that bind to it most strongly. This is what we will do in this project. When we have read all the sequences we will assemble them into a vast table using the same computing techniques that scientists use to understand the human genome. This table will tell us how sequences can be linked together to make an aptamer that sticks to molecules as tightly as an antibody. By making aptamers that stick as tightly as antibodies we will break down the barrier that is preventing their other advantages from being used. The will lead to new and improved tests that allow scientists and physicians to detect many different kinds of molecule in the same minute spot of blood, and new drugs that seek out and destroy cancer cells and harmful viruses.

At present antibodies are the main class of recognition molecule for non-nucleic acid target molecules. Aptamers are alternatives to antibodies that have many potential advantages. Up until now they have been identified by the SELEX process which involves multiple cycles of in vitro selection and amplification. A literature review shows that in general aptamers identified by SELEX have lower affinities than antibodies. This shortcoming is a barrier to their more widespread use. Recent work has shown that aptamer affinities can be increased by linking them to sequences that are eliminated in SELEX. This suggests that the affinities of aptamers would be improved if such sequences could be identified. The main aim of the proposed work is to show that aptamers with improved affinities can be identified by one-step in vitro selection and massively parallel DNA sequencing. In Step 1 a random library of approximately 1015 oligonucleotides will be incubated with a protein target molecule attached to magnetic beads. In Step 2 the 105 sequences that bound most strongly to the target will be clonally amplified on hydrogel micro-beads. In Step 3 the beads will be loaded into a multiwell plate, and in Step 4 the oligonucleotides attached to the beads will be sequenced. In Step 5 the sequences will be assembled to produce a table of aligned base frequencies. This table will be based on thousands of sequences and therefore differences in affinity contributed by individual bases will be evident from their higher frequency at a given locus. Importantly this table will contain information about sequences that contribute to affinity, but which would be eliminated in SELEX. Consensus sequences derived from the table will be validated by comparison with aptamers identified by SELEX. A secondary aim of the work is to show how clonally amplified beads attached to millions of potential aptamer sequences can be interrogated for functions other than simple binding to a target molecule.

The main areas in which improved aptamers will have an impact are diagnostics, therapy and separation technologies. The three main groups of beneficiaries will be producers of improved aptamers, direct-users of improved aptamers and end-users of products and services based on improved aptamers. Producers of aptamers are organisations that would supply aptamers to direct users such as academics and specialised diagnostic companies. Alta Bioscience (see attached letter of support) is a potential producer of improved aptamers. Producer organisations will benefit economically from the sale of aptamers to direct users. These economic benefits will create skilled jobs in the UK and internationally, and benefit Liverpool University by payment of licensing fees. The time-scale for producers to benefit from improved aptamers will depend on demand, but we anticipate that at least one producer will commit to the production of improved aptamers within a year of the research project's conclusion. Direct users of improved aptamers are organisations that would use aptamers to improve existing applications or develop new ones. Examples of direct users are academics engaged in proteomics and commercial organisations developing diagnostic devices such as BBI (see attached letter of support). Some direct users such as Noxxon (a developer of therapeutic aptamers - see attached letter of support) would produce improved aptamers in-house, but others such as academics are expected to obtain them from producers. Direct users will also include public bodies such as Dstl (part of the UK Ministry of Defence - see attached letter of support) and Fera (UK Food and Environmental Research Agency - see attached letter of support) who would use improved aptamers to develop field-portable biosensor devices. The activities of all direct-users will support skilled jobs and create new ones, while in-house production of improved aptamers by organisations such as Noxxon will benefit Liverpool University by payment of licensing fees. The time-scale for direct-users to benefit from improved aptamers will depend on how quickly these can be incorporated into products, but we anticipate that the first of these based would be developed within two-to-three years from the end of the project. Academics at Liverpool University and other institutions will benefit from these further developments in the form research contracts and consultancies. End-users of improved aptamers are organisations and individuals who will benefit from the activities and/or products of direct-users. One example of an end-user group is physicians who will benefit from the improved knowledge gained by academics engaged in proteomics and from the provision of new and more affordable tests supplied by diagnostic companies such as ValiRx, Oxford Gene Technology and Proteome Sciences (see attached letters of support). Other examples of end-user groups are public bodies such as law-enforcement agencies and environmental protection agencies that will benefit from field-portable tests developed by direct-users such as BBI, Dstl and Fera. All these activities will lead to improvements in health-care, the environment and security, that will benefit the general public in the UK and internationally.